Systems and methods for endoscopic angle-resolved low coherence interferometry

ABSTRACT

Fourier domain a/LCI (faLCI) system and method which enables in vivo data acquisition at rapid rates using a single scan. Angle-resolved and depth-resolved spectra information is obtained with one scan. The reference arm can remain fixed with respect to the sample due to only one scan required. A reference signal and a reflected sample signal are cross-correlated and dispersed at a multitude of reflected angles off of the sample, thereby representing reflections from a multitude of points on the sample at the same time in parallel. Information about all depths of the sample at each of the multitude of different points on the sample can be obtained with one scan on the order of approximately 40 milliseconds. From the spatial, cross-correlated reference signal, structural (size) information can also be obtained using techniques that allow size information of scatterers to be obtained from angle-resolved data.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/725,603 entitled “SYSTEMS AND METHODS FOR ENDOSCOPICANGLE-RESOLVED LOW COHERENCE INTERFEROMETRY,” filed on Oct. 11, 2005,which is incorporated herein by reference in its entirety.

This application is also related to U.S. Pat. No. 7,102,758 entitled“FOURIER DOMAIN LOW-COHERENCE INTERFEROMETRY FOR LIGHT SCATTERINGSPECTROSCOPY APPARATUS AND METHOD,” which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

Fourier domain angle-resolved low coherence interferometry (faLCI)system and method that enables data acquisition of angle-resolved anddepth-resolved spectra information of a sample, in which depth and sizeinformation about the sample can be obtained with a single scan at rapidrates for in vivo applications in particular.

BACKGROUND OF THE INVENTION

Examining the structural features of cells is essential for manyclinical and laboratory studies. The most common tool used in theexamination for the study of cells has been the microscope. Althoughmicroscope examination has led to great advances in understanding cellsand their structure, it is inherently limited by the artifacts ofpreparation. The characteristics of the cells can only been seen at onemoment in time with their structure features altered because of theaddition of chemicals. Further, invasion is necessary to obtain the cellsample for examination.

Thus, light scattering spectrography (LSS) was developed to allow for invivo examination applications, including cells. The LSS techniqueexamines variations in the elastic scattering properties of cellorganelles to infer their sizes and other dimensional information. Inorder to measure cellular features in tissues and other cellularstructures, it is necessary to distinguish the singly scattered lightfrom diffuse light, which has been multiply scattered and no longercarries easily accessible information about the scattering objects. Thisdistinction or differentiation can be accomplished in several ways, suchas the application of a polarization grating, by restricting or limitingstudies and analysis to weakly scattering samples, or by using modelingto remove the diffuse component(s).

As an alternative approach for selectively detecting singly scatteredlight from sub-surface sites, low-coherence interferometry (LCI) hasalso been explored as a method of LSS. LCI utilizes a light source withlow temporal coherence, such as broadband white light source forexample. Interference is only achieved when the path length delays ofthe interferometer are matched with the coherence time of the lightsource. The axial resolution of the system is determined by the coherentlength of the light source and is typically in the micrometer rangesuitable for the examination of tissue samples. Experimental resultshave shown that using a broadband light source and its second harmonicallows the recovery of information about elastic scattering using LCI.LCI has used time depth scans by moving the sample with respect to areference arm directing the light source onto the sample to receivescattering information from a particular point on the sample. Thus, scantimes were on the order of 5-30 minutes in order to completely scan thesample.

Angle-resolved LCI (a/LCI) has been developed as a means to obtainsub-surface structural information regarding the size of a cell. Lightis split into a reference and sample beam, wherein the sample beam isprojected onto the sample at different angles to examine the angulardistribution of scattered light. The a/LCI technique combines theability of (LCI) to detect singly scattered light from sub-surface siteswith the capability of light scattering methods to obtain structuralinformation with sub-wavelength precision and accuracy to constructdepth-resolved tomographic images. Structural information is determinedby examining the angular distribution of the back-scattered light usinga single broadband light source is mixed with a reference field with anangle of propagation. The size distribution of the cell is determined bycomparing the osciallary part of the measured angular distributions topredictions of Mie theory. Such a system is described in CellularOrganization and Substructure Measured Using Angle-ResolvedLow-Coherence Inteferometry, Biophysical Journal, 82, April 2002,2256-2265, incorporated herein by reference in its entirety.

The a/LCI technique has been successfully applied to measuring cellularmorphology and to diagnosing intraepithelial neoplasia in an animalmodel of carcinogenesis. The inventors of the present applicationdescribed such a system in Determining nuclear morphology using animproved angle-resolved low coherence interferometry system in OpticsExpress, 2003, 11(25): p. 3473-3484, incorporated herein by reference inits entirety. The a/LCI method of obtaining structural information abouta sample has been successfully applied to measuring cellular morphologyin tissues and in vitro as well as diagnosing intraepithelial neoplasiaand assessing the efficacy of chemopreventive agents in an animal modelof carcinogenesis. a/LCI has been used to prospectively grade tissuesamples without tissue processing, demonstrating the potential of thetechnique as a biomedical diagnostic.

Initial prototype and second generation a/LCI systems required 30 and 5minutes respectively to obtain similar data. These earlier systemsrelied on time domain depth scans just as provided in previous LCI basedsystems. The length of the reference arm of the interferometer had to bemechanically adjusted to achieve serial scanning of the detectedscattering angle. The method of obtaining angular specificity wasachieved by causing the reference beam of the interferometry scheme tocross the detector plane at a variable angle. This general method forobtaining angle-resolved, depth-resolved backscattering distributionswas disclosed in U.S. Pat. No. 6,847,456 entitled “Methods and systemsusing field-based light scattering spectroscopy,” which is incorporatedby reference herein in its entirety.

Other LCI prior systems are disclosed in U.S. Pat. Nos. 6,002,480 and6,501,551, both of which are incorporated by reference herein in theirentireties. U.S. Pat. No. 6,002,780 covers obtaining depth-resolvedspectroscopic distributions and discusses obtaining the size ofscatterers by observing changes in wavelength due to elastic scatteringproperties. U.S. Pat. No. 6,501,551 covers endoscopic application ofinterferometric imaging and does anticipate the use of Fourier domainconcepts to obtain depth resolution. U.S. Pat. No. 6,501,551 does notdiscuss measurement of angularly resolved scattering distributions, theuse of scattered light to determine scatterer size by analysis ofelastic scattering properties, nor the use of an imaging spectrometer torecord data in parallel, whether that data is scattering or imagingdata. Finally, U. S. Pat. No. 7,061,622 discusses fiber optic means formeasuring angular scattering distributions, but does not discuss theFourier domain concept. Also because it describes an imaging technique,the embodiments all include focusing optics which limit the regionprobed.

SUMMARY OF THE INVENTION

The present invention involves a new a/LCI technique called Fourierdomain a/LCI (faLCI), which enables data acquisition at rapid ratesusing a single scan, sufficient to make in vivo applications feasible.The present invention obtains angle-resolved and depth-resolved spectrainformation about a sample, in which depth and size information aboutthe sample can be obtained with a single scan, and wherein the referencearm can remain fixed with respect to the sample due to only one scanrequired. A reference signal and a reflected sample signal arecross-correlated and dispersed at a multitude of reflected angles off ofthe sample, thereby representing reflections from a multitude of pointson the sample at the same time in parallel.

Since this angle-resolved, cross-correlated signal is spectrallydispersed, the new data acquisition scheme is significant as it permitsdata to be obtained in less than one second, a threshold determined tobe necessary for acquiring data from in vivo tissues. Information aboutall depths of the sample at each of the multitude of different points onthe sample can be obtained with one scan on the order of approximately40 milliseconds. From the spatial, cross-correlated reference signal,structural (size) information can also be obtained using techniques thatallow size information of scatterers to be obtained from angle-resolveddata.

The faLCI technique of the present invention uses the Fourier domainconcept to acquire depth resolved information. Signal-to-noise andcommensurate reductions in data acquisition time are possible byrecording the depth scan in the Fourier (or spectral) domain. The faLCIsystem combines the Fourier domain concept with the use of an imagingspectrograph to spectrally record the angular distribution in parallel.Thereafter, the depth-resolution of the present invention is achieved byFourier transforming the spectrum of two mixed fields with theangle-resolved measurements obtained by locating the entrance slit ofthe imaging spectrograph in a Fourier transform plane to the sample.This converts the spectral information into depth-resolved informationand the angular information into a transverse spatial distribution. Thecapabilities of faLCI have been initially demonstrated by extracting thesize of polystyrene beads in a depth-resolved measurement.

Various mathematical techniques and methods are provided for determiningsize information of the sample using the angle-resolved,cross-correlated signal.

The present invention is not limited to any particular arrangement. Inone embodiment, the apparatus is based on a modified Mach-Zehnderinterferometer, wherein broadband light from a superluminescent diode issplit into a reference beam and an input beam to the sample by abeamsplitter. In another embodiment, a unique optical fiber probe can beused to deliver light and collect the angular distribution of scatteredlight from the sample of interest.

The a/LCI method can be a clinically viable method for assessing tissuehealth without the need for tissue extraction via biopsy or subsequenthistopathological evaluation. The a/LCI system can be applied for anumber of purposes: early detection and screening for dysplasticepithelial tissues, disease staging, monitoring of therapeutic actionand guiding the clinician to biopsy sites. The non-invasive,non-ionizing nature of the optical a/LCI probe means that it can beapplied frequently without adverse affect. The potential of a/LCI toprovide rapid results will greatly enhance its widespread applicabilityfor disease screening.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The accompanying drawing figures incorporated in and forming a part ofthis specification illustrate several aspects of the invention, andtogether with the description serve to explain the principles of theinvention.

FIG. 1A is a schematic of one exemplary embodiment of the faLCI systememploying Mach-Zehnder interferometer;

FIG. 1B is an illustration showing the relationship of the detectedscattering angle to slit of spectrograph in the interferometerarrangement of FIG. 1A;

FIG. 2 is a flowchart illustrating the steps performed by theinterferometer apparatus to recover depth-resolved spatialcross-correlated information about the sample for analysis;

FIGS. 3A-D illustrate examples of faLCI data recovered in the spectraldomain for an exemplary sample of polystyrene beads, comprising thetotal acquired signal (FIG. 3A), the reference field intensity (FIG.3B), the signal field intensity (FIG. 3C), and the extracted,cross-correlated signal between the reference and signal fieldintensities (FIG. 3D);

FIG. 4A is an illustration of the axial spatial cross-correlatedfunction performed on the cross-correlated faLCI data illustrated inFIG. 3D as a function of depth and angle;

FIG. 4B is an illustration of an angular distribution plot of raw andfiltered data regarding scattered sample signal intensity as a functionof angle in order to recover size information about the sample;

FIG. 5A is an illustration of the filtered angular distribution of thescattered sample signal intensity compared to the best fit Mie theory todetermine size information about the sample;

FIG. 5B is a Chi-squired minimization of size information about thesample to estimate the diameter of cells in the sample;

FIG. 6 is a schematic of exemplary embodiment of the faLCI systememploying an optical fiber probe;

FIG. 7A is a cutaway view of an a/LCI fiber-optic probe tip that may beemployed by the faLCI system illustrated in FIG. 6;

FIG. 7B illustrates the location of the fiber probe in the faLCI systemillustrated in FIG. 7A;

FIG. 8A is an illustration of an alternative fiber-optic faLCI systemthat may be employed with the present invention;

FIG. 8B is an illustration of sample illumination and scattered lightcollection with distal end of probe in the faLCI system illustrated inFIG. 8B; and

FIG. 8C is an illustration of an image of the illuminated distal end ofprobe of the faLCI system illustrated in FIG. 8A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments set forth below represent the necessary information toenable those skilled in the art to practice the invention and illustratethe best mode of practicing the invention. Upon reading the followingdescription in light of the accompanying drawing figures, those skilledin the art will understand the concepts of the invention and willrecognize applications of these concepts not particularly addressedherein. It should be understood that these concepts and applicationsfall within the scope of the disclosure and the accompanying claims.

The present invention involves a new a/LCI technique called Fourierdomain a/LCI (faLCI), which enables data acquisition at rapid ratesusing a single scan, sufficient to make in vivo applications feasible.The present invention obtains angle-resolved and depth-resolved spectrainformation about a sample, in which depth and size information aboutthe sample can be obtained with a single scan, and wherein the referencearm can remain fixed with respect to the sample due to only one scanrequired. A reference signal and a reflected sample signal arecross-correlated and dispersed at a multitude of reflected angles off ofthe sample, thereby representing reflections from a multitude of pointson the sample at the same time in parallel.

Since this angle-resolved, cross-correlated signal is spectrallydispersed, the new data acquisition scheme is significant as it permitsdata to be obtained in less than one second, a threshold determined tobe necessary for acquiring data from in vivo tissues. Information aboutall depths of the sample at each of the multitude of different points onthe sample can be obtained with one scan on the order of approximately40 milliseconds. From the spatial, cross-correlated reference signal,structural (size) information can also be obtained using techniques thatallow size information of scatterers to be obtained from angle-resolveddata.

The faLCI technique of the present invention uses the Fourier domainconcept to acquire depth resolved information. Signal-to-noise andcommensurate reductions in data acquisition time are possible byrecording the depth scan in the Fourier (or spectral) domain. The faLCIsystem combines the Fourier domain concept with the use of an imagingspectrograph to spectrally record the angular distribution in parallel.Thereafter, the depth-resolution of the present invention is achieved byFourier transforming the spectrum of two mixed fields with theangle-resolved measurements obtained by locating the entrance slit ofthe imaging spectrograph in a Fourier transform plane to the sample.This converts the spectral information into depth-resolved informationand the angular information into a transverse spatial distribution. Thecapabilities of faLCI have been initially demonstrated by extracting thesize of polystyrene beads in a depth-resolved measurement.

The key advances of the present invention can be broken down into threecomponents: (1) new rapid data acquisition methods, (2) fiber probedesigns, and (3) data analysis schemes. Thus, the present invention isdescribed in this matter for convenience in its understanding.

An exemplary apparatus, as well as the steps involved in the process ofobtaining angle and depth-resolved distribution data scattered from asample, are also set forth in FIG. 2. The faLCI scheme in accordancewith one embodiment of the present invention is based on a modifiedMach-Zehnder interferometer as illustrated in FIG. 1A. Broadband light10 from a superluminescent diode (SLD) 12 is directed by a mirror 13(step 60 in FIG. 2) and split into a reference beam 14 and an input beam16 to a sample 18 by beamsplitter BSI 20 (step 62 in FIG. 3). The outputpower of the SLD 12 may be 3 milliwatts, having a specification ofλo=850 nm, Δλ=20 nm FWHM for example, providing sufficiently lowcoherence length to isolate scattering from a cell layer within tissue.The path length of the reference beam 14 is set by adjustingretroreflector RR 22, but remains fixed during measurement. Thereference beam 14 is expanded using lenses L1 (24) and L2 (26) to createillumination (step 64 in FIG. 2), which is uniform and collimated uponreaching a spectrograph slit 48 in an imaging spectrograph 29. Forexample, L1 may have a focal length of 1.5 centimeters, and L2 26 mayhave focal length of 15 centimeters.

Lenses L3 (31) and L4 (38) are arranged to produce a collimated pencilbeam 30 incident on the sample 18 (step 66 in FIG. 2). By displacinglens L4 (38) vertically relative to lens L3 (31), the input beam 30 ismade to strike the sample at an angle of 0.10 radians relative to theoptical axis. This arrangement allows the full angular aperture of lensL4 (38) to be used to collect scattered light 40 from the sample 18.Lens L4 (38) may have a focal length of 3.5 centimeters.

The light 40 scattered by the sample 18 is collected by lens L4 (32) andrelayed by a 4f imaging system comprised of lenses L5 (43) and L6 (44)such that the Fourier plane of lens L4 (32) is reproduced in phase andamplitude at the spectrograph slit 48 (step 68 in FIG. 2). The scatteredlight 40 is mixed with the reference field 14 at a second beamsplitterBS2 42 with the combined fields 46 falling upon the entrance slit(illustrated in FIG. 1B as element 48) to the imaging spectrograph 29(step 70 in FIG. 2). The imaging spectrograph 29 may be the modelSP2150i, manufactured by Acton Research for example. FIG. 1B illustratesthe distribution of scattering angle across the dimension of the slit48. The mixed fields are dispersed with a high resolution grating (e.g.1200 l/mm) and detected using a cooled CCD 50 (e.g. 1340×400, 20 μm×20μm pixels, Spec10:400, manufactured by Princeton Instruments) (step 72in FIG. 2).

The detected signal 46 is a function of vertical position on thespectrograph slit 48, y, and wavelength λ once the light is dispersed bythe spectrograph 29. The detected signal at pixel (m, n) can be relatedto the signal 40 and reference fields 16 (E_(s), E_(r)) as:I(λ_(m) , y _(n))=<|E _(r)(λ_(m) , y _(n))|²>+<|E_(s)(λ_(m) , y_(n))|²>+2Re<E _(s)(λ_(m) , y _(n))E _(r)*(λ_(m) , y _(n))>cos φ,   (1)where φ is the phase difference between the two fields 30, 16 and < . .. > denotes an ensemble average in time. The interference term isextracted by measuring the intensity of the signal 30 and referencebeams 16 independently and subtracting them from the total intensity.

In order to obtain depth resolved information, the wavelength spectrumat each scattering angle is interpolated into a wavenumber (k=2π/λ)spectrum and Fourier transformed to give a spatial cross correlation,Γ_(SR)(z) for each vertical pixel y_(n):Γ_(SR)(z,y _(n))=∫dk e ^(ikz) <E _(s)(k, y _(n))E _(r)*(k,y _(n))>cosφ  (2)The reference field 14 takes the form:E _(r)(k)=E _(o) exp└−((k−k _(o))/Δk)²┘exp└−((y−y_(o))/Δy)²┘exp[ikΔl]  (3)where k_(o) (y_(o) and Δk (Δy) represent the center and width of theGaussian wavevector (spatial) distribution and Δl is the selected pathlength difference. The scattered field 40 takes the formE _(s)(k, θ)=Σ_(j) E _(o) exp[−((k−k _(o))/Δk)²]exp[ikl _(j) ]S _(j)(k,θ)   (4)where S_(j) represents the amplitude distribution of the scatteringoriginating from the jth interface, located at depth I_(j). The angulardistribution of the scattered field 40 is converted into a positiondistribution in the Fourier image plane of lens L4 through therelationship y=f₄θ. For the pixel size of the CCD 50 (e.g. 20 μm), thisyields an angular resolution (e.g. 0.57 mrad) and an expected angularrange (e.g. 228 mrad.).

Inserting Eqs. (3) and (4) into Eq. (2) and noting the uniformity of thereference field 14 (Δy>>slit height) yields the spatial crosscorrelation at the nth vertical position on the detector 29:$\begin{matrix}{{\Gamma_{SR}\left( {z,y_{n}} \right)} = {\sum\limits_{j}{\int{{\mathbb{d}k}{E_{o}}^{2}{\exp\left\lbrack {{- 2}\left( {{\left( {k - k_{o}} \right)/\Delta}\quad k} \right)^{2}} \right\rbrack}{\exp\left\lbrack {{\mathbb{i}}\quad{k\left( {z - {\Delta\quad l} + l_{j}} \right)}} \right\rbrack} \times {S_{j}\left( {k,{\theta_{n} = {y_{n}/f_{4}}}} \right)}\cos\quad{\phi.}}}}} & (5)\end{matrix}$Evaluating this equation for a single interface yields:Γ_(SR)(z, y _(n))=|E _(n)|² exp[−((z−Δl+l _(j))Δk)²/8[S _(j)(k_(o),θ_(n) =y _(n) /f ₄)cos φ.   (6)

Here we have assumed that the scattering amplitude S does not varyappreciably over the bandwidth of the source light 12. This expressionshows that we obtain a depth resolved profile of the scatteringdistribution 40 with each vertical pixel corresponding to a scatteringangle.

FIG. 3A below shows typical data representing the total detectedintensity (Equation (1), above) of the sum of the reference field 16 andthe field scattered 40 by a sample of polystyrene beads, in thefrequency domain given as a function of wavelength and angle, given withrespect to the backwards scattering direction. In an exemplaryembodiment, this data was acquired in 40 milliseconds and records dataover 186 mrad, approximately 85% of the expected range, with some lossof signal at higher angles.

FIGS. 3B and 3C illustrate the intensity of the reference and signalfields 14, 30 respectively. Upon subtraction of the signal and referencefields 14, 30 from the total detected intensity, the interference 46between the two fields is realized as illustrated in FIG. 3D. At eachangle, interference data 46 are interpolated into k-space and Fouriertransformed to give the angular depth resolved profiles of the sample 18as illustrated in FIG. 4A. The Fourier transform of the angle-resolved,cross correlated signal 46, which is the result of signal 40 scatteredat a multitude of reflected angles off the sample 18 and obtained in theFourier plane of lens L4 (32), produces depth-resolved information aboutthe sample 18 as a function of angle and depth. This providesdepth-resolved information about the sample 18. Because theangle-resolved, cross-correlated signal 46 is spectrally dispersed, thedata acquisition permits data to be obtained in less than one second.Information about all depths of the sample 18 at each of the multitudeof different points (i.e. angles) on the sample 18 can be obtained withone scan on the order of approximately 40 milliseconds. Normally, timedomain based scanning is required to obtain information about all depthsof a sample at a multitude of different points, thus requiringsubstantial time and movement of the reference arm with respect to thesample.

In the experiments that produced the depth-resolved profit of the sample18 illustrated in FIG. 4A, the sample 18 consists of polystyrenemicrospheres (e.g. n=1.59, 10.1 μm mean diameter, 8.9% variance, NISTcertified, Duke Scientific) suspended in a mixture of 80% water and 20%glycerol (n=1.36) to provide neutral buoyancy. The solution was preparedto obtain a scattering length I=200 μm. The sample is contained in around well (8 mm diameter, 1 mm deep) behind a glass coverslip(thickness, d˜170 μm) (not shown). The sample beam 30 is incident on thesample 18 through the coverslip. The round trip thickness through thecoverslip (2 n d=2 (1.5) (170 μm)=0.53 mm—see FIG. 4A) shows the depthresolved capability of the approach. The data are ensemble averaged byintegrating over one mean free path (MFP). The spatial average canenable a reduction of speckle when using low-coherence light to probe ascattering sample. To simplify the fitting procedure, the scatteringdistribution is low pass filtered to produce a smoother curve, with thecutoff frequency chosen to suppress spatial correlations on lengthscales above 16 μm.

In addition to obtaining depth-resolved information about the sample 18,the scattering distribution data (i.e. a/LCI data) obtained from thesample 18 using the disclosed data acquisition scheme can also be usedto make a size determination of the nucleus using the Mie theory. Ascattering distribution 74 of the sample 18 is illustrated in FIG. 4B asa contour plot. The raw scattered information 74 about the sample 18 isshown as a function of the signal field 30 and angle. A filtered curveis determined using the scattered data 74. Comparison of the filteredscattering distribution curve 76 (i.e. a representation of the scattereddata 74) to the prediction of Mie theory (curve 78 in FIG. 5A) enables asize determination to be made.

In order to fit the scattered data 76 to Mie theory, the a/LCI signalsare processed to extract the oscillatory component which ischaracteristic of the nucleus size. The smoothed data 76 are fit to alow-order polynomial (4^(th) order was used for example herein, butlater studies use a lower 2^(nd) order), which is then subtracted fromthe distribution 76 to remove the background trend. The resultingoscillatory component is then compared to a database of theoreticalpredictions obtained using Mie theory 78 from which the slowly varyingfeatures were similarly removed for analysis.

A direct comparison between the filtered a/LCI data 76 and Mie theorydata 78 may not possible, as the chi-squared fitting algorithm tends tomatch the background slope rather than the characteristic oscillations.The calculated theoretical predictions include a Gaussian distributionof sizes characterized by a mean diameter (d) and standard deviation(δD) as well as a distribution of wavelengths, to accurately model thebroad bandwidth source.

The best fit (FIG. 5A) is determined by minimizing the Chi-squaredbetween the data 76 and Mie theory (FIG. 5B), yielding a size of10.2±1.7 μm, in excellent agreement with the true size. The measurementerror is larger than the variance of the bead size, most likely due tothe limited range of angles recorded in the measurement.

As an alternative to processing the a/LCI data and comparing to Mietheory, there are several other approaches which could yield diagnosticinformation. These include analyzing the angular data using a Fouriertransform to identify periodic oscillations characteristic of cellnuclei. The periodic oscillations can be correlated with nuclear sizeand thus will possess diagnostic value. Another approach to analyzinga/LCI data is to compare the data to a database of angular scatteringdistributions generated with finite element method (FEM) or T-Matrixcalculations. Such calculations may offer superior analysis as there arenot subject to the same limitations as Mie theory. For example, FEM orT-Matrix calculations can model non-spherical scatterers and scattererswith inclusions while Mie theory can only model homogenous spheres.

As an alternative embodiment, the present invention can also employoptical fibers to deliver and collect light from the sample of interestto use in the a/LCI system for endoscopic applications. This alternativeembodiment is illustrated in FIG. 6.

The fiber optic a/LCI scheme for this alternative embodiment makes useof the Fourier transform properties of a lens. This property states thatwhen an object is placed in the front focal plane of a lens, the imageat the conjugate image plane is the Fourier transform of that object.The Fourier transform of a spatial distribution (object or image) isgiven by the distribution of spatial frequencies, which is therepresentation of the image's information content in terms of cycles permm. In an optical image of elastically scattered light, the wavelengthretains its fixed, original value and the spatial frequencyrepresentation is simply a scaled version of the angular distribution ofscattered light.

In the fiber optic a/LCI scheme, the angular distribution is captured bylocating the distal end of the fiber bundle in a conjugate Fouriertransform plane of the sample using a collecting lens. This angulardistribution is then conveyed to the distal end of the fiber bundlewhere it is imaged using a 4f system onto the entrance slit of animaging spectrograph. A beamsplitter is used to overlap the scatteredfield with a reference field prior to entering the slit so that lowcoherence interferometry can also be used to obtain depth resolvedmeasurements.

Turning now to FIG. 6, the fiber optic faLCI scheme is shown. Light 12′from a broadband light source 10′ is split into a reference field 14′and a signal field 16′ using a fiber splitter (FS) 80. A splitter ratioof 20:1 is chosen in one embodiment to direct more power to a sample 18′via the signal arm 82 as the light returned by the tissue is typicallyonly a small fraction of the incident power.

Light in the reference fiber 14′ emerges from fiber F1 and is collimatedby lens L1 (84) which is mounted on a translation stage 86 to allowgross alignment of the reference arm path length. This path length isnot scanned during operation but may be varied during alignment. Acollimated beam 88 is arranged to be equal in dimension to the end 91 offiber bundle F3 (90) so that the collimated beam 88 illuminates allfibers in F3 with equal intensity. The reference field 14′ emerging fromthe distal tip of F3 (90) is collimated with lens L3 (92) in order tooverlap with the scattered field conveyed by fiber F4 (94). In analternative embodiment, light emerging from fiber F1 (14′) is collimatedthen expanded using a lens system to produce a broad beam.

The scattered field is detected using a coherent fiber bundle. Thescattered field is generated using light in the signal arm 82 which isdirected toward the sample 18′ of interest using lens L2 (98). As withthe free space system, lens L2 (98) is displaced laterally from thecenter of single-mode fiber F2 such that a collimated beam is producedwhich is traveling at an angle relative to the optical axis The factthat the incident beam strikes the sample at an oblique angle isessential in separating the elastic scattering information from specularreflections. The light scattered by the sample 18′ is collected by afiber bundle consisting of an array of coherent single mode ormulti-mode fibers. The distal tip of the fiber is maintained one focallength away from lens L2 (98) to image the angular distribution ofscattered light. In the embodiment shown in FIG. 6, the sample 18′ islocated in the front focal plane of lens L2 (98) using a mechanicalmount 100. In the endoscope compatible probe shown in FIG. 7, the sampleis located in the front focal plane of lens L2 (98) using a transparentsheath (element 102).

As illustrated in FIG. 6 and also FIG. 7B, scattered light 104 emergingfrom a proximal end 105 of the fiber probe F4 (94) is recollimated bylens L4 (104) and overlapped with the reference field 14′ usingbeamsplitter BS (108). The two combined fields 110 are re-imaged ontothe slit (element 48′ in FIG. 7) of the imaging spectrograph 29′ usinglens L5 (112). The focal length of lens L5 (112) may be varied tooptimally fill the slit 48′. The resulting optical signal containsinformation on each scattering angle across the vertical dimension ofthe slit 48′ as described above for the apparatus of FIGS. 1A and 1B.

It is expected that the above-described a/LCI fiber-optic probe willcollect the angular distribution over a 0.45 radian range (approx. 30degrees) and will acquire the complete depth resolved scatteringdistribution 110 in a fraction of a second.

There are several possible schemes for creating the fiber probe whichare the same from an optical engineering point of view. One possibleimplementation would be a linear array of single mode fibers in both thesignal and reference arms. Alternatively, the reference arm 96 could becomposed of an individual single mode fiber with the signal arm 82consisting of either a coherent fiber bundle or linear fiber array.

The fiber probe tip can also have several implementations which aresubstantially equivalent. These would include the use of a drum or balllens in place of lens L2 (98). A side-viewing probe could be createdusing a combination of a lens and a mirror or prism or through the useof a convex mirror to replace the lens-mirror combination. Finally, theentire probe can be made to rotate radially in order to provide acircumferential scan of the probed area.

Yet another data acquisition embodiment of the present invention couldbe a fa/LCI system is based on a modified Mach-Zehnder interferometer asillustrated in FIG. 5A. The output 10″ from a fiber-coupledsuperluminescent diode (SLD) source 12″ (e.g. Superlum, P_(o)=15 mW,λo=841.5 nm, Δλ=49.5 nm, coherence length=6.3 μm) is split into samplearm delivery fiber 16″ and a reference arm delivery fiber 14″ by a 90/10fiber splitter FS (80″) (e.g. manufactured by AC Photonics). The samplearm delivery fiber 16″ can consist of either of the following forexample: (1) a single mode fiber with polarization control integrated atthe tip; or (2) a polarization maintaining fiber. A sample probe 113 isassembled by affixing the delivery fiber 16″(NA≅0.12 ) along the ferrule114 at the distal end of a fiber bundle 116 such that the end face ofthe delivery fiber 16″ is parallel to and flush with the face of thefiber bundle 116. Ball lens L1 (115) (e.g. f_(l)=2.2 mm) is positionedone focal length from the face of the probe 113 and centered on thefiber bundle 116, offsetting the delivery fiber 16″ from the opticalaxis of lens L1 (115). This configuration, which is also depicted inFIG. 8B, produces a collimated beam 120 (e.g. P=9 mW) with a diameter(e.g. 2f₁NA) of 0.5 mm incident on the sample 18″ at an angle of 0.25rad. for example.

The scattered light 122 from the sample is collected by lens L1 (115)and, via the Fourier transform property of the lens L1 (115, the angulardistribution of the scattered field 122 is converted into a spatialdistribution at the distal face of the multimode coherent fiber bundle116 (e.g. Schott North America, Inc., length=840 mm, pixel size=8.2 □m,pixel count=13.5K) which is located at the Fourier image plane of lensL1 (115). The relationship between vertical position on the fiberbundle, y′, and scattering angle, θ is given by y′=f₁θ. As anillustration, the optical path of light scattered 122 at three selectedscattering angles is shown in FIG. 8B. Overall, the angular distributionis sampled by approximately 130 individual fibers for example, across avertical strip of the fiber bundle 116″, as depicted by the highlightedarea in FIG. 8C. The 0.2 mm, for example, thick ferrule (d₁) separatingthe delivery fiber 16″ and fiber bundle 116 limits the minimumtheoretical collection angle (θ_(min,th)=d₁/f₁) to 0.09 rad in thisexample. The maximum theoretical collection angle is determined by d₁and d₂, the diameter of the fiber bundle, by θ_(max,th)=(d₁+d₂)/f₁ to be0.50 rad.

Experiments using a standard scattering sample 122 indicate the usableangular range to be θ_(min)=0.12 rad. to θ_(max)=0.45 rad. d_(1,), forexample, can be minimized by fabricating a channel in the distal ferrule123 and positioning the delivery fiber 16″ the channel. The fiber bundle116 is spatially coherent, resulting in a reproduction of the collectedangular scattering distribution at the proximal face. Additionally, asall fibers in the bundle 116 are path length matched to within thecoherence length, the optical path length traveled by scattered light122 at each angle is identical. The system disclosed in“Fiber-optic-bundle-based optical coherence tomography,” by T. Q. Xie,D. Mukai, S. G. Guo, M. Brenner, and Z. P. Chen in Optics Letters30(14), 1803-1805 (2005) (hereinafter “Xie”), incorporated by referenceherein in its entirety, discloses a multimode coherent fiber bundle intoa time-domain optical coherence tomography system and demonstrated thatthe modes of light coupled into an individual fiber will traveldifferent path lengths. In the example herein of the present invention,it was experimentally determined that the higher order modes are offsetfrom the fundamental mode by 3.75 mm, well beyond the depth (˜100 μm)required for gathering clinically relevant data. Additionally, the powerin the higher order modes had a minimal affect on dynamic range as thesample arm power is significantly less than the reference arm power.Finally, it should be noted that while the system disclosed in Xiecollected data serially through individual fibers, the example of thepresent invention herein uses 130 fibers to simultaneously collectscattered light across a range of angles in parallel, resulting in rapiddata collection.

The angular distribution exiting a proximal end 124 of the fiber bundle116 is relayed by the 4f imaging system of L2 and L3 (f₂=3.0 cm,f₃=20.0cm) to the input slit 48″ of the imaging spectrograph 29″ (e.g. ActonResearch, InSpectrum 150). The theoretical magnification of the 4fimaging system is (f₃/f₂) 6.67 in this example. Experimentally, themagnification was measured to be M=7.0 in this example with thediscrepancy most likely due to the position of the proximal face 124 ofthe fiber bundle 116 with relation to lens L2 (126). The resultingrelationship between vertical position on the spectrograph slit 48″, y,and θ is y=Mf₁(θ−θ_(min)). The optical path length of the reference armis matched to that of the fundamental mode of the sample arm. Light 127exiting the reference fiber 14″ is collimated by lens L4 (128) (e.g.f=3.5 cm, spot size=8.4 mm) to match the phase front curvature of thesample light and to produce even illumination across the slit 48″ of theimaging spectrograph 29″. A reference field 130 may be attenuated by aneutral density filter 132 and mixed with the angular scatteringdistribution at beamsplitter BS (134). The mixed fields 136 aredispersed with a high resolution grating (e.g. 1200 lines/mm) anddetected using an integrated, cooled CCD (not shown) (e.g. 1024×252, 24μm×24 μm pixels, 0.1 nm resolution) covering a spectral range of 99 nmcentered at 840 nm, for example.

The detected signal 136, a function of wavelength, λ, and θ, can berelated to the signal and reference fields (Es, Er) as:I(λ_(m),θ_(n))=<|E _(r)(λ_(m),θ_(n))|² >+<|E _(s)(λ_(m),θ_(n))|²>+2Re<E_(s)(λ_(m), θ_(n))E _(r)*(λ_(m),θ_(n))cos(φ)>,   (1)where φ is the phase difference between the two fields, (m,n) denotes apixel on the CCD, and < . . . > denotes a temporal average. I(λ_(m),θ_(n)) is uploaded to a PC using LabVIEW manufactured by NationalInstruments software and processed in 320 ms to produce a depth andangle resolved contour plot of scattered intensity. The processing ofthe angle-resolved scattered field to obtain depth and size informationdescribed above, and in particular reference to the data acquisitionapparatus of FIGS. 1A and 1B, can then used to obtain angle-resolved,depth-resolved information about the sample 18″ using the scatteredmixed field 136 generated by the apparatus in FIG. 8.

The embodiments set forth above represent the necessary information toenable those skilled in the art to practice the invention and illustratethe best mode of practicing the invention. Upon reading the followingdescription in light if the accompanying drawings figures, those skilledin the art will understand the concepts of the invention and willrecognize applications of these concepts not particularly addressedherein. It should be understood that these concepts and applicationsfall within the scope of the disclosure.

Those skilled in the art will recognize improvements and modificationsto the preferred embodiments of the present invention. All suchimprovements and modifications are considered within the scope of theconcepts disclosed herein and the claims that follow.

1. A method of obtaining depth-resolved spectra of a sample fordetermining depth characteristics of scatterers within the sample,comprising the steps of: emitting a source beam onto a splitter, whereinthe splitter is fixed with respect to the sample, and wherein thesplitter splits light from the source beam to produce a reference beamand a sample beam; directing the sample beam towards the sample at anangle; receiving an angle-resolved reflected sample beam as a result ofthe sample beam reflectively scattering at a multitude of reflectedangles off of the sample in parallel at the same time, wherein theangle-resolved reflected sample beam contains the angular scatteringdistribution of the reflected sample beam; cross-correlating theangled-resolved reflected sample beam with the reference beam to producean angle-resolved cross-correlated signal about the sample; spectrallydispersing the angle-resolved cross-correlated signal to yield a single,angle-resolved, spectrally-resolved cross-correlation reflection profilehaving depth-resolved information about the sample at each of themultitude of reflected angles; and Fourier transforming of theangled-resolved cross-correlated signal to produce depth-resolvedinformation about the sample as a function of angle and depth.
 2. Themethod of claim 1, further comprising processing the angle-resolvedcross-correlated signal to obtain depth-resolved information about thescatterers of the sample at a multitude of different points on thesample from the angle-resolved, spectrally-resolved cross-correlationreflection profile.
 3. The method of claim 1, further comprisingrecovering size information about the scatterers from theangle-resolved, spectrally-resolved cross-correlation reflectionprofile.
 4. The method of claim 3, wherein recovering the sizeinformation is obtained by comparing the angular scattering distributionof the sample to a predicted analytically or numerically calculatedangular scattering distribution of the sample.
 5. The method of claim 4,wherein the predicted analytically or numerically calculated angularscattering distribution of the sample is a Mie theory angular scatteringdistribution of the sample.
 6. The method of claim 4, further comprisingfiltering the angular scattering distribution of the sample before thestep of comparing.
 7. The method of claim 4, further comprisingcalculating a Gaussian distribution of sizes of the scatterers bycalculating a mean diameter and a standard deviation to model theangular scattering distribution of the sample.
 8. The method of claim 1,further comprising the step of collimating the sample beam to produce acollimated sample beam, wherein the step of directing the sample beamtowards the sample at an angle comprises directing the collimated samplebeam towards the sample at an angle.
 9. The method of claim 1, furthercomprising the step of collimating the reference beam to produce acollimated reference beam.
 10. The method of claim 1, wherein thereference beam is reflected before the step of cross-correlating tocreate a reflected reference beam.
 11. The method of claim 10, whereinthe reflected reference beam is created by reflecting the reference beamoff of a reference mirror.
 12. The method of claim 1, wherein thesplitter is an optical fiber splitter.
 13. The method of claim 1,wherein the source beam is comprised of a light selected from the groupconsisting of a white light from an arc lamp, a thermal source, a LED, acoherent light from a broadband laser, a superluminescent diode, a diodelaser, and a supercontinuum source.
 14. The method of claim 1, whereinthe step of cross-correlating the angle-resolved reflected sample beamwith the reference beam comprises: determining an interference term bymeasuring the intensity of the angle-resolved reflected sample beam andthe reference beam independently; and subtracting the interference termfrom the total intensity of the angle-resolved reflected sample beam.15. The method of claim 1, wherein the length of the path of thereference beam is fixed.
 16. The method of claim 1, wherein the splitteris attached to a fixed reference arm.
 17. The method of claim 1, whereinthe sample is attached to a fixed sample arm.
 18. The method of claim 1,wherein the step of spectrally dispersing the angle-resolvedcross-correlated signal comprises directing the angle-resolved reflectedsample beam which has been combined with the reference beam into aspectrograph.
 19. The method of claim 18, wherein the spectrographcomprises an imaging spectrograph comprised of a plurality of imagingpoints wherein each of the plurality of imaging points corresponds to aspecific scattering angle in order to produce the angle-resolved,spectrally-resolved cross-correlation reflection profile about thesample.
 20. The method of claim 18, wherein the spectrograph comprises amulti-channel spectrograph comprised of a plurality of channels, whereineach of the plurality of channels corresponds to a specific scatteringangle in order to produce the angle-resolved, spectrally-resolvedcross-correlation reflection profile about the sample.
 21. The method ofclaim 1, wherein the step of receiving the angle-resolved reflectedsample beam as a result of the sample beam reflectively scattering atthe multitude of reflected angles off of the sample in parallel at thesame time comprises capturing the angular distribution of the reflectedsample beam at an end of a fiber bundle comprised of a plurality offibers.
 22. The method of claim 21, wherein the plurality of fibers inthe fiber bundle are arranged to collect different angular reflectionsof the reflected sample beam to collect the angular scatteringdistribution of the reflected sample beam.
 23. The method of claim 21,wherein the fiber bundle comprises a linear array of single mode fibers.24. The method of claim 21, further comprising the step of carrying thesample beam on a delivery fiber wherein the delivery fiber delivers thesample beam at an oblique angle with respect to the sample and the fiberbundle so that the specular reflection due to the sample is not receivedby the fiber bundle.
 25. The method claim 21, further comprising thestep of receiving the angle-resolved reflected sample beam via a Fouriertransform property of an optical element placed in between thefiber-optic bundle and the sample to receive the angle-resolvedreflected sample beam located at the other focus of the optical element.26. The method of claim 21, wherein the plurality of fibers possess thesame or substantially the same spatial arrangement at distal andproximal ends of the plurality of fibers so that the fiber bundle isspatially coherent with respect to conveying the angular distribution ofthe angle-resolved reflected sample beam.
 27. The method of claim 1,further comprising the step of splitting more light at the splitter fromthe source beam to produce more light in the sample beam than in thereference beam.
 28. The method of claim 25 wherein the optical elementis either a lens or an imaging optical element.
 29. An apparatus forobtaining depth-resolved spectra of a sample for determining size anddepth characteristics of scatterers within a sample, comprising: areceiver that is fixed with respect to the sample that: receives anangle-resolved reflected sample beam to produce, via a Fouriertransform, depth-resolved information about the sample as a function ofangle and depth, created as a result of a sample beam, split by asplitter from a source beam, which is reflectively scattering at amultitude of reflected angles off of the sample, in parallel at the sametime, wherein the angle-resolved reflected sample beam contains theangular scattering distribution of the angle-resolved reflected samplebeam; receives a reference beam split by the splitter from the sourcebeam; and cross-correlates the angle-resolved reflected sample beam withthe reference beam to produce an angle-resolved and depth-resolvedcross-correlated signal about the sample; a detector that spectrallydisperses the angle-resolved and depth-resolved cross-correlated signalto yield a single, angle-resolved, spectrally-resolved cross-correlationreflection profile having depth-resolved information about the sample ateach of the multitude of reflected angles; and a processor that receivesthe single, angle-resolved, spectrally-resolved cross-correlationreflection profile.
 30. The apparatus of claim 29, wherein the processoris adapted to determine the depth of the scatterers of the sample at amultitude of different points on the sample from the single,angle-resolved, spectrally-resolved cross-correlation reflectionprofile.
 31. The apparatus of claim 29, wherein the processor is adaptedto recover size information about the scatterers from theangle-resolved, spectrally-resolved cross-correlation reflectionprofile.
 32. The apparatus of claim 31, wherein the processor recoversthe size information by comparing the angular scattering distribution ofthe sample to a predicted analytically or numerically calculated angularscattering distribution of the sample.
 33. The apparatus of claim 32,wherein the predicted analytically or numerically calculated angularscattering distribution of the sample is a Mie theory angular scatteringdistribution of the sample.
 34. The apparatus of claim 32, wherein theprocessor is further adapted to filter the angular scatteringdistribution of the sample before comparing the angular scatteringdistribution of the sample to a predicted analytically or numericallycalculated angular scattering distribution of the sample.
 35. Theapparatus of claim 34, wherein the processor is further adapted toidentify a Gaussian distribution of sizes of the scatterers byidentifying a mean diameter and a standard deviation to model theangular scattering distribution.
 36. The apparatus of claim 29, whereinthe sample beam is collimated.
 37. The apparatus of claim 29 wherein thereference beam is collimated.
 38. The apparatus of claim 29, furthercomprising a reflection device to receive and reflect the reference beamwherein the receiver receives the reflected reference beam.
 39. Theapparatus of claim 38, wherein the reflection device is a referencemirror.
 40. The apparatus of claim 29, wherein the splitter is anoptical fiber splitter.
 41. The apparatus of claim 29, wherein thesource beam is comprised of a light selected from the group consistingof a white light from an arc lamp, a thermal source, a LED, a coherentlight from a broadband laser, a superluminescent diode, a diode laser,and a supercontinuum source.
 42. The apparatus of claim 29, wherein thelength of the path of the reference beam is fixed.
 43. The apparatus ofclaim 29, wherein the splitter is attached to a fixed reference arm. 44.The apparatus of claim 29, wherein the sample is attached to a fixedsample arm.
 45. The apparatus of claim 29, further comprising aspectrograph that receives the angle-resolved reflected sample beam andthe reference beam to produce the angle-resolved and depth-resolvedcross-correlated signal about the sample.
 46. The apparatus of claim 29,wherein the receiver is a fiber bundle comprised of a plurality offibers.
 47. The apparatus of claim 46, wherein the plurality of fibersin the fiber bundle are arranged to collect different angularreflections of the reflected sample beam to collect the angularscattering distribution of the angle-resolved reflected sample beam. 48.The apparatus of claim 46, wherein the fiber bundle comprises a lineararray of single mode fibers.
 49. The apparatus of claim 47, furthercomprising a delivery fiber that carries the sample beam so that thedelivery fiber delivers the sample beam at an oblique angle with respectto the sample and the fiber bundle so that the specular reflection dueto the sample is not received by the fiber bundle.
 50. The apparatus ofclaim 47, wherein the plurality of fibers is positioned at one focus ofan optical element to receive the angle-resolved reflected sample beamwhich is located at the other focus of the optical element such that thefiber bundle receives the angular distribution of scattered light via aFourier transform property of the optical element.
 51. The apparatus ofclaim 47, wherein the plurality of fibers possess the same orsubstantially the same spatial arrangement at distal and proximal endsof the plurality of fibers so that the fiber bundle is spatiallycoherent with respect to conveying the angular distribution of theangle-resolved reflected sample beam.
 52. The apparatus of claim 29,wherein the splitter splits more light from the source beam to producethe source beam than to produce the reference beam.
 53. The apparatus ofclaim 50 wherein the optical element is either a lens or an imagingoptical element.
 54. An apparatus for obtaining depth-resolved spectraof a sample for determining size and depth characteristics of scattererswithin a sample, comprising: a delivery fiber that carries a sample beamwherein the sample beam is directed to the sample over the deliveryfiber and scattered at a multitude of reflected angles off of the sampleto produce a scattered sample beam; a fiber-optic bundle receivercomprised of a plurality of fibers positioned at one focus of an opticalelement to receive the scattered sample beam reflected by the samplewhich is located at the other focus of the optical element, such thatthe fiber-optic bundle receiver receives the angular distribution of thescattered sample beam via a Fourier transform property of the opticalelement. a detector that spectrally disperses the scattered sample beamto yield an angle-resolved, spectrally-resolved reflection profile ateach of the multitude of reflected angles in parallel at the same time;and a processor that receives and analyzes the angle-resolved,spectrally-resolved reflection profile.
 55. The apparatus of claim 54,wherein the plurality of fibers in the fiber-optic bundle receiver arearranged to collect different angular reflections of the sample beam tocollect the angular scattering distribution of the scattered samplebeam.
 56. The apparatus of claim 54, wherein the fiber-optic bundlereceiver comprises a linear array of single mode fibers.
 57. Theapparatus of claim 54, wherein the plurality of fibers possess the samespatial arrangement at distal and proximal ends of the plurality offibers so that the fiber-optic bundle receiver is spatially coherentwith respect to conveying the angular scattering distribution ofscattered sample beam.
 58. The apparatus of claim 54 wherein the opticalelement is either a lens or an imaging optical element.